Alveolar macrophages play a critical role in the pathogenesis of inhalational anthrax: phagocytosing anthrax endospores, and transporting them to the mediastinal lymph nodes where the spores germinate and release toxins. Although macrophages are capable of killing the spores within their phagolysosomes, this innate immune response often fails to prevent germination and outgrowth of bacilli - resulting in fatal anthrax. We propose that the efficiency of killing of ingested endospores is a crucial determinant of the clinical consequences from inhalational exposure. Our hypothesis is that B. anthracis has evolved a mechanism to exploit the normal phagosomal processing, thereby avoiding destruction within the phagolysosome. We propose to first analyze the mechanisms used by B. anthracis endospores to survive within human alveolar macrophages. These studies will determine the efficiency of endospore killing by human macrophages, the ability of B. anthracis toxins or cell wall/capsule components to interfere with endospore killing, and the functional maturation of B. anthracis containing phagosomes. Next we will assess the consequences of B. anthracis infection on the activation/deactivation status of human alveolar macrophages, focusing on the synthesis of cytokines and the generation of reactive oxygen and nitrogen intermediates. Finally, we will analyze the effect of lethal toxin on the human alveolar macrophage. The impact of lethal toxin-mediated cleavage of mitogen-activated protein kinase kinase isoforms will be determined, then additional potential targets of lethal toxin-mediated cleavage will be explored using a proteomic and reverse genetics approach. While there is abundant evidence that alveolar macrophages play a critical role in inhalational anthrax in mice, many details remain uncertain. Furthermore, nothing is known regarding human alveolar macrophages and anthrax. In close collaboration with the other projects in this program, this project will define the interactions between human alveolar macrophages and anthrax spores, and analyze how the macrophage's defense is breached. These studies should thus provide the information needed to begin to develop new strategies to protect the public against possible bioterror associated anthrax exposure.